| 1.Object Pogostemon cablin was selected as our research subject,of which the impurities,moisture,total ash,acid-insoluble ash,alcohol-soluble extracts and leaf percentages specified in the Pharmacopoeia were detected.At the same time,the contents of volatile oil,heavy metals and harmful elements,pesticide residues,aflatoxin and so on are well defined;the thin-layer chromatography(TLC)was employeed to identify the different habitats of the patchouli and Agastache rugosa;multi-components approach by single marker(QAMS)for gas chromatography(GC)was established to simultaneously determine four components in patchouli and investigate the effect of habitats and leaf percentages on the content of patchouli.Fingerprinting of the patchouli was studied by GC,and the quality relationship between the producing areas and the percentage of patchouli leaves on the intrinsic quality of patchouli was overall evaluated,and the quality of patchouli was comprehensively assessed.We hope to provide reference for the further study of the quality control of patchouli.2 Methods 2.1 Collection of Pogostemon cablin and study on its inspection items In this study,50 batches of Pogostemon cablin were collected,and the leaf percentages of each batch of patchouli were measured.The 2015 Pharmacopoeia defines the limits of impurities,moistuer,total ash,acid-insoluble ash and alcohol-soluble extracts of the patchouli.In this assay,all the patchouli were tested including the above items.The contents of volatile oil,heavy metals and harmful elements,pesticide residues and aflatoxin in patchouli were further measured and the limitation regulations were furmulated.2.2 TLC identification of Pogostemon cablin By investigating the extraction method,eluting solvent,thin layer plate,temperature,humidity and sample size,a thin-layer chromatography method was established to simutaneously detect two constituents in patchouli,patchouli alcohol and pogostone.The method can be utilized to distinguish Pogostemon cablin from Agastache rugosa.2.3 Determination of four components in Pogostemon cablin by GC quantitative analysis of multi-components by single marker The GC quantitative analysis of multi-components by single marker method was established to simultaneously determine the four components of patchouli.By calculating the relative correction factors(RCF)of patchouli alcohol and the other three components,the content of patchouli alcohol was calculated by internal standard method,and the RCF was used to calculate the contents of ?-elemene,?-caryophyllene,and pogostone,and the methodological investigation,system suitability and durability were investigated.The calculated value was compared with the value measured by the conventional internal standard method by t-test analysis of variance method to discuss the feasibility of QAMS.The effects of the different habitats and leaf percentages on the content of the four components of the Pogostemon cablin were studied.2.4 Study on GC Fingerprints of Pogostemon cablin The fingerprints of 50 batches of patchouli from different habitats were analyzed by GC,and the methodological investigation was carried out.Using the software of “Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica(2012 edition)” to evaluate the similarity.The effects of the different habitats and leaf percentages on the overall quality of the patchouli medicinal materials were studied.3 results 3.1 Collection of Pogostemon cablin and study on its inspection items The impurities,moisture,total ash,acid-insoluble ash,alcohol-soluble extracts,leaf percentages were detected.The results are in consistency with the 2015 edition of the Chinese Pharmacopoeia.The results of heavy metal determination showed that the most samples have qualified metal content requied by the standard regulation.The results of nine organochlorine pesticide residues pesticide residues such as carbendazim showed that the total contents in all samples meet the 2341 General Principles requirement of the Chinese Pharmacopoeia in 2015.The results of aflatoxin assay showed that the content of aflatoxin B1,B2,G1 and G2 are below the limitation of the total amount of aflatoxin(10 ?g/kg).3.2 TLC identification of Pogostemon cablin A TLC method for simultaneous detection of patchouli alcohol and pogostone was established.The experimental durability of temperature and humidity showed that the separation effect,stability and safety of the method were optimal.The thin-layer chromatographic conditions can distinguish Pogostemon cablin from Agastache rugosa.3.3 Determination of four components in Pogostemon cablin by GC quantitative analysis of multi-components by single marker The RCF calculated in this experiment are of good reproducibility and can be used as a stable parameter in the calculation of ?-elemene,?-caryophyllene,and pogostone in patchouli.The methodological and system durability results showed that the content determination method of patchouli with patchouli alcohol as the control met the quantitative analysis requirements.The contents of 50 batches of patchouli were determined by this method.Compared with the results of conventional internal standard method,there was no significant difference.This method is suitable for the simultaneous determination of four components of patchouli alcohol,?-elemene,?-caryophyllene and pogostone.Analysis of the four components' contents in patchouli demonstrated: the ratio of leaf was the main factor affecting the content of four components of patchouli.3.4 Study on GC Fingerprints of Pogostemon cablin The established GC fingerprint has good precision,repeatability and stability.The results of the proportion of common peak areas in different habitats showed that the similarity of each producing area is above 0.9.The study on the area of the common peaks of Pogostemon cablin with different leaf percentage showed that the common peaks area increases with the increase of leaf percentage,indicating that leaf percentage was the main factor affecting the quality of patchouli.4 Conclusion In this paper,the Pogostemon cablin was used as our research object,and the inspection items specified in the pharmacopoeia were tested.At the same time,some limitation inspection items were supplemented to improve the quality evaluation of patchouli.A TLC method for simultaneous determination of two components of patchouli alcohol and pogostone was established,which can distinguish Pogostemon cablin from Agastache rugosa.A QAMS method was established to simultaneously determine the four components in the patchouli,and the feasibility was verified.The GC fingerprint of the patchouli was contructed.The overall evaluation on patchouli from different habitats and with different leaf percentages indicated there's an apparent correlation between the quality of patchouli and its leaf percentage.The study can lay a foundation for improving the quality and clinical application of patchouli. |
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